For the realization of metrological jobs on and in biological liquids such as water samples or food, detection methods based on biological receptor molecules may be employed. A metrological job may consist in detecting minimum quantities of an analyte in a solution or sample, which makes tremendous demands on measurement technology. In these methods, receptor molecules ensure the sensitivity and selectivity of the detection (of an analyte or an analyte concentration) by reacting with the analyte and, for example, binding therewith (e.g. via an antibody-antigene interaction). For detecting this reaction, conversion to a processable (optical or electrical) signal is necessitated. This may be effected by means of a physical signal converter—the so-called transducer—for example. A respective biosensor therefore operates on the principle that first a biological or biochemical reaction occurs and then this reaction is converted to a measurable signal.
One major challenge in the development of biosensors consists in coupling the biological receptor molecules and the physical signal converter such that there is maximum and efficient signal carry between the two. This means, for example, that even minimum quantities of the substance to be detected (analyte) (e.g. down to 10−13 mol/L for tumor markers in the blood serum or 1 . . . 30 mmol/L for glucose concentrations) will result in a measurable signal.
For effecting a reaction between the receptor molecules and the analyte in the sample to be detected, the receptor molecules are first immobilized. For the immobilization of biomolecules, hydrogels and aerogels, for example, may be used in biosensor technology and chromatography. On the other hand, known optical transducers are based on light formed by optical elements (open beam) and, apart from that, on guiding light through an optical waveguide. Light formed by the optical elements may be utilized for transmission measurements, for example, or may be guided along optical trains in the style of microscopy or fluorescence microscopy. The optical waveguide may serve two functions:
(1) transporting light to the location where the analysis takes place or
(2) providing an evanescent electromagnetic field on the surface of the waveguide so that the electromagnetic field interacts with the biomolecules found there or the reactions executing there.
The presence of analyte molecules may be evidenced optically as follows:
(i) by interaction with dielectric properties of the molecules on the surface of the waveguide and evidence of a resulting phase shift in an optical interferrometer,
(ii) via selective decoupling of certain waveguide modes from the waveguide due to the refractive index of analyte molecules,
(iii) via a specific absorption of a molecule (e.g., interaction of an analyte molecule with a receptor molecule may manifest itself in a formation of a specific spectral line),
(iv) via a fluorescence of the molecules or the marker fluorophores attached to the molecules,
(v) via fluorescence quenching as a result of specific reactions (e.g., a selected fluorescence may be quenched when the receptor molecules interact or bind with the analyte molecules),
(vi) via a resonant energy transfer between fluorescent molecules (FRET) and the changes thereof in dependence on the analyte concentration.
The optical waveguide transducers have in common that they either have to in part make do with unguided light and the resulting diffraction and aperture effects in the interaction with the medium to be examined, or that only a small portion of the light (the evanescent field) achieves interaction with the analyte and/or receptor molecules. In order to eliminate the latter problem, above all materials having a very high refractive index (e.g. Ta2O5, TiO2) are used, of which very thin and well-controlled layers may be fabricated in complex, clean-room-based processes. The thin layers may range below 100 nm or slightly above.
For the aforementioned reasons, the number of receptor molecules capable of taking part in evidencing molecules is strictly limited. Open-beam optical solutions, in spite of being very efficient, suffer from the high space requirements of good beam-forming elements (such as lenses, mirrors, etc.). The use of microlenses provides only little improvement as the microlenses in turn strictly limit the measurable sample volume. Particularly in the field of microfluidic biosensors and biochips in but also in the field of real-time sensors suitable for long-term use, the issues mentioned pose substantial limitations.